Molecular diagnosis of apple virus and viroid pathogens from India

Apple is known to be susceptible to various virus and viroid pathogens. Symptomatic apple cultivars and rootstocks were collected and analyzed by ELISA and then through RT-PCR. The study reports the presence of Apple mosaic virus (ApMV), Apple stem grooving virus (ASGV), Apple stem pitting virus (ASPV), Apple chlorotic leaf spot virus (ACLSV), the major apple viruses and Prunus necrotic ringspot virus (PNRSV), a minor apple virus, at the molecular level in India. Apple scar skin viroid (ASSVd) infection was also confirmed at the molecular level. Sporadic incidences of Tomato ringspot virus and Arabis mosaic virus infections were also detected by ELISA in nursery plants.     

Global Changes in the Mammary Epigenome Are Induced by Hormonal Cues and Coordinated by Ezh2

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Highlights? The mammary epigenome is highly sensitive to steroid hormones at specific developmental stages ? Ezh2 links hormonal cues to changes in chromatin structure and gene expression ? Progesterone is a key in vivo regulator of Ezh2 ? Hormone-induced chromatin changes likely play a role in the initiation of breast cancer     

Fluorescence Adherence Inhibition Assay: A Novel Functional Assessment of Blocking Virus Attachment by Vaccine-Induced Antibodies

Neutralizing antibodies induced by vaccination or natural infection play a critically important role in protection against the viral diseases. In general, neutralization of the viral infection occurs via two major pathways: pre- and post-attachment modes, the first being the most important for such infections as influenza and polio, the latter being significant for filoviruses. Neutralizing capacity of antibodies is typically evaluated by virus neutralization assays that assess reduction of viral infectivity to the target cells in the presence of functional antibodies. Plaque reduction neutralization test, microneutralization and immunofluorescent assays are often used as gold standard virus neutralization assays. However, these methods are associated with several important prerequisites such as use of live virus requiring safety precautions, tedious evaluation procedure and long assessment time. Hence, there is a need for a robust, inexpensive high throughput functional assay that can be performed rapidly using inactivated virus, without extensive safety precautions. Herein, we report a novel high throughput Fluorescence Adherence Inhibition assay (fADI) using inactivated virus labeled with fluorescent secondary antibodies virus and Vero cells or erythrocytes as targets. It requires only few hours to assess pre-attachment neutralizing capacity of donor sera. fADI assay was tested successfully on donors immunized with polio, yellow fever and influenza vaccines. To further simplify and improve the throughput of the assay, we have developed a mathematical approach for calculating the 50% titers from a single sample dilution, without the need to analyze multi-point titration curves. Assessment of pre- and post-vaccination human sera from subjects immunized with IPOL^®, YF-VAX^® and 2013–2014 Fluzone^® vaccines demonstrated high efficiency of the assay. The results correlated very well with microneutralization assay performed independently by the FDA Center of Biologics Evaluation and Research, with plaque reduction neutralization test performed by Focus Diagnostics, and with hemaglutination inhibition assay performed in-house at Sanofi Pasteur. Taken together, fADI assay appears to be a useful high throughput functional immunoassay for assessment of antibody-related neutralization of the viral infections for which pre-attachment neutralization pathway is predominant, such as polio, influenza, yellow fever and dengue.     

Studies on Enhancement of Anti-microbial Activity of Pristine MWCNTs Against Pathogens

Carbon nanotubes (CNTs), owing to their inherently unique properties in the domain of biomedical sciences including drug delivery, offer an exciting platform to the researchers. Of late, their applications have also been successfully established. Recently, single-walled CNTs (SWCNTs) have been explored for antibacterial efficacy, but naïve multi-walled CNTs (MWCNTs) still remained unearthed. The present studies endeavor the investigation of the potential of various non-ionic surfactants in solubility enhancement of MWCNTs and their subsequent antibacterial efficacy against Escherichia coli and Staphylococcus aureus. Polysorbates offer more solubility to MWCNTs vis-à-vis the phospholipids. However, the antibacterial effect was found to be less influenced by solubility but significantly determined by the type of surfactant. Transmission electron photomicrographs confirmed significant adhesion of MWCNTs to the bacterial walls only in the presence of unsaturated phospholipids and this was expressed in the form of lowest minimum inhibitory concentration (MIC) values of MWCNTs dispersed with the same. The findings are unique as MWCNTs were found to be active against both Gram-negative and Gram-positive bacteria to a similar extent, though somewhat milder than SWCNTs. However, when dispersed with unsaturated phospholipids, the former offer almost comparable antibacterial effects to that of the latter. The study opens a new research domain to further explore the antibacterial effects of non-functionalized and relatively safer MWCNTs, accentuating the importance of biocomponents like unsaturated phospholipids in this purview.     

QbD-Oriented Development and Characterization of Effervescent Floating-Bioadhesive Tablets of Cefuroxime Axetil

The objective of the present studies was systematic development of floating-bioadhesive gastroretentive tablets of cefuroxime axetil employing rational blend of hydrophilic polymers for attaining controlled release drug delivery. As per the QbD-based approach, the patient-centric target product profile and quality attributes of tablet were earmarked, and preliminary studies were conducted for screening the suitability of type of polymers, polymer ratio, granulation technique, and granulation time for formulation of tablets. A face-centered cubic design (FCCD) was employed for optimization of the critical material attributes, i.e., concentration of release controlling polymers, PEO 303 and HPMC K100 LV CR, and evaluating in vitro buoyancy, drug release, and ex vivo mucoadhesion strength. The optimized formulation was embarked upon through numerical optimization, which yield excellent floatation characteristic with drug release control (i.e., T _60%?>?6 h) and bioadhesion strength. Drug-excipient compatibility studies through FTIR and P-XRD revealed the absence of any interaction between the drug and polymers. In vivo evaluation of the gastroretentive characteristics through X-ray imaging and in vivo pharmacokinetic studies in rabbits revealed significant extension in the rate of drug absorption (i.e., T _max, K _a, and MRT) from the optimized tablet formulation as compared to the marketed formulation. Successful establishment of various levels of in vitro/in vivo correlations (IVIVC) substantiated high degree of prognostic ability of in vitro dissolution conditions in predicting the in vivo performance. In a nutshell, the studies demonstrate successful development of the once-a-day gastroretentive formulations of cefuroxime axetil with controlled drug release profile and improved compliance.     

Response surface methodology for lovastatin production by Aspergillus terreus GD13 strain

A wild type Aspergillus terreus GD13 strain, chosen after extensive screening, was optimized for lovastatin production using statistical Box-Behnken design of experiments. The interactive effect of four process parameters, i.e. lactose and soybean meal, inoculum size (spore concentration) and age of the spore culture, on the production of lovastatin was evaluated employing response surface methodology (RSM). The model highlighted the positive effect of soybean meal concentration and inoculum level for achieving maximal level of lovastatin (1342 mg/l). The optimal fermentation conditions improved the lovastatin titre by 7.0-folds when compared to the titres obtained under unoptimized conditions.